Polyethylene glycols ("PEGs") are long chain, linear synthetic polymers composed of ethylene oxide units. The ethylene oxide units can vary such that PEG compounds can be obtained with molecular weights ranging from approximately 200-100,000. PEG typically is colorless, odorless, soluble in water, stable to heat, does not hydrolyze or deteriorate, and is non-toxic. As such, PEGs have been extensively studied for use in pharmaceuticals, on artificial implants, and other applications where biocompatibility is of importance.
The attachment of PEG to proteins ("PEGylation") has been shown to: (1) increase the plasma half-life of the proteins, see e.g., U.S. Pat. No. 5,349,052, Delgado et al., issued Sep. 20, 1994; (2) alter the biochemical and physical properties of the protein, including increasing solubility, Chen et al., Biocem. Biophys. Acta, 660: 293-298 (1988), and improving protection against proteolysis, Sada, et al., J. Fermentation Bioengineering 71: 137-139 (1991); and (3) reduce antigenicity, Davis et al., "Biomedical Polymers. Polymeric Materials and Pharmaceuticals for Biomedical Use", (Eds. Goldberg & Nakajima) pp 441-452, Academic Press, N.Y. (1980).
Various derivatives of PEG have been proposed that have an active moiety for permitting PEG to be attached to proteins. The vast majority of PEGylating reagents react with free primary amino groups of the polypeptide. Most of these free amines are the .epsilon.-NH.sub.2 group of lysine amino acid residues; and, because typical proteins possess a large number of lysine residues, random attachment of multiple PEG molecules often occurs, which can lead to loss of protein activity.
Published PCT Publication No. WO 95/13312 describes, inter alia, water soluble sulfone-activiated PEGs which are highly selective for coupling with thiol moieties instead of amino moieties on molecules and on surfaces. These PEG derivatives are stable against hydrolysis for extended periods in aqueous environments at pHs of about 11 or less, and can form linkages with molecules to form conjugates which are also hydrolytically stable. The linkage by which the PEGs and the biologically active molecule are coupled includes a sulfone moiety coupled to a thiol moiety and has the structure PEG-SO.sub.2 -CH.sub.2 -CH.sub.2 -S-W, where W represents the biologically active molecule, and wherein the sulfone moiety is vinyl sulfone or an active ethyl sulfone.
One example of a biologically active molecule which can be modified by such sulfone-activated polymers is TNF binding protein ("TNFbp")(also referred to as TNF inhibitor). TNFbp is of therapeutic interest in the treatment of disease states such as rheumatoid arthritis and septic shock. Several forms of native TNFbp, e.g., 30 kDa and 40 kDa TNFbp have been described in detail; see e.g., published European Patent Application No. 90 113 673.9, incorporated herein by reference. Furthermore, various TNFbp muteins, e.g., c105 30 kDa TNFbp mutein (the asparagine residue at position 105 of the native human protein is changed to cysteine) have been prepared and described; see published PCT Publication No. WO 92/16221, incorporated herein by reference.
PCT Publication No. WO 92/16221 further describes TNFbp forms represented by the formula R.sub.1 -X-R.sub.2 (referred to as a "dumbbell", compound), wherein R.sub.1 and R.sub.2 are biologically active TNFbp groups and X is a non-peptidic polymeric spacer with at least one Michael acceptor group, e.g. a sulfone-activated PEG. One particular compound, referred to as c105 TNFbp mutein dumbbell, is a homodumbbell compound wherein R.sub.1 and R.sub.2 is a c105 30 kDa TNFbp mutein, X is 20 K polyethylene glycol bis-vinylsulfone (20 K PEGbv), and wherein R.sub.1 and R.sub.2 are attached site-specifically at the cysteine 105 residue to the PEGbv. This c105 TNFbp mutein dumbbell was reported to be many times more active than the unPEGylated native 30 kDa TNFbp, unPEGylated c105 30 kDa TNFbp mutein or the PEGylated 30 kDa TNFbp "monobell" (R.sub.1 -X).
A problem commonly encountered by those skilled in the art when producing compounds such as the c105 TNFbp mutein dumbbell is that the procedures used to synthesize the 20 K PEGbv, or other polymer, have several drawbacks, depending on the synthesis procedure used. One particularly problematic drawback is that the resultant 20 K PEGbv will often contain high molecular weight PEG impurities and PEG mono-vinylsulfone. These impurities can then adversely affect the overall yield and purity of the desired dumbbell compound. The need exists, therefore, for methods which would further purify the PEGbv, or other polymers, and thereby enhance the overall purity and yield of the desired dumbbell compound, and make utilization of such compounds as therapeutic agents more commercially practicable.
A number of groups have reported on the use of reversed-phase high performance liquid chromatography (rpHPLC) to separate low- and medium-molecular weight (&lt;5K) PEGs on the basis of size; see e.g., Murphy et al., J. Chromat., 211: 160-165 (1981); Lai et al., J. High Res. Chrom. & Chrom. Comm., 7: 494-496 (1984); Barka & Hoffman, J. Chromat., 389: 273-278 (1987). These techniques, while relatively effective, suffer from their use of expensive resins and organic solvents. Also, these methods are analytical rather than preparative procedures. There have been no reports of successful separation of larger molecular weight PEGs using HPLC. Medium- and high-molecular-weight (1K-40K) PEGs have been analyzed by size exclusion chromatography on a variety of derivatized silica supports; Engelhardt & Mathes, J. Chromat., 185: 305 (1979).
Hydrophobic interaction chromatography (HIC) has achieved acceptance as an analytical and preparative purification method for biomolecules. The chemical principles underlying HIC are similar to those involved in salt precipitation, and related to those associated with reversed phase liquid chromatography (RPLC), in that all operate on the basis of hydrophobicity. HIC is characterized by adsorption of molecules to a weakly hydrophobic surface at high salt concentrations, followed by elution with a decreasing salt gradient. Thus, HIC combines the non-denaturing characteristics of salt precipitation and the precision of chromatography to yield excellent activity recoveries. Moreover, because HIC operates at lower binding energy, it does not require the use of expensive organic solvents in the mobile phase.
To date, there have been no reports concerning the use of HIC resins to separate PEGs, especially high molecular weight PEGs.